1. Field of the Invention
The invention relates to novel semaphorins which are distinguished by a particular domain structure and derivatives thereof, nucleic acids (DNA, RNA, cDNA) which code for these semaphorins, and derivatives thereof, and the preparation and use thereof.
2. Description of the Related Art
The publications which are referenced in this application describe the state of the art to which this invention pertains. These references are incorporated herein by references.
Semaphorins were described for the first time by Kolodkin {Kolodkin et al. (1993) Cell 75:1389-1399} as members of a conserved gene family.
The genes or parts of the genes of other semaphorins have now been cloned and, in some cases, characterized. To date, a total of 5 human (H-Sema III, H-Sema V, H-Sema IV, H-SemaB and H-SemaE) {Kolodkin et al. (1993); Roche et al. (1996) Onkogene 12:1289-1297; Sekido et al. (1996) Proc. Natl. Acad. Sci. USA 93:4120-4125; Xiang et al. (1996) Genomics 32:39-48; Hall et al. (1996) Proc. Natl. Acad. Sci. USA 39:11780-11785; Yamada et al. (1997) (GenBank Accession No. AB000220)}, 8 murine (mouse genes; M-Sema A to M-Sema-H) {Püschel et al. (1995) Neuron 14:941-948; Messerschmidt et al. (1995) Neuron 14:949-959; Inigaki et al. (1995) FEBS Letters 370:269-272; Adams et al. (1996) Mech. Dev. 57:3345; Christensen et al. (1996)(GenBank Accession No. Z80941, Z93948)}, 5 galline (chicken) (collapsin-1 to -5) {Luo et al. (1993); Luo et al. (1995) Neuron 14:1131-1140}, and genes from rats (R-Sema-III) {Giger et al. (1996) J. Comp. Neurol. 375:378-392}, zebra fish, insects (fruit fly (Drosophila melanogaster: D-Sema I and D-Sema II), beetles (Tribolium confusum: T-Sema-I), grasshoppers (Schistocerca americana: G-Sema-I)) {Kolodkin et al. (1993)}, and nematodes (C. elegans: Ce-Sema) {Roy et al. (1994) (GenBank Accession No. U15667)} have been disclosed. In addition, two poxviruses (vaccinia (ORF-A39) and variola (ORFA39-homologous)) {Kolodkin et al. (1993)} and alcelaphine herpesvirus Type 1 (AHV-1) (AHV-Sema) {Ensser and Fleckenstein (1995) Gen. Virol. 76:1063-1067} have genes homologous to semaphorins.
Table 1 summarizes the semaphorins identified to date in various species. Table 1 indicates the names of the semaphorins (column 1), the synonyms used (column 2), the species from which the particular semaphorin has been isolated (column 3) and, where known, data on the domain structure of the encoded protein and on the chromosomal location (column 4 in Table 1), the accession number under which the sequence of the gene is stored in gene databanks (for example in an EST (expressed sequence tags) databank, EMBL (European Molecular Biology Laboratory, Heidelberg) or NCBI (National Center for Biotechnology Information, Maryland, USA), and the corresponding reference under which these data have been published (column 5 in Table 1).
All the gene products (encoded semaphorins) of the semaphorin genes disclosed to date have an N-terminal signal peptide which has at its C-terminal end a characteristic Sema domain with a length of about 450 to 500 amino acids. Highly conserved amino acid motifs and a number of highly conserved cysteine residues are located within the Sema domains. The gene products (semaphorins) differ in the C-terminal sequences which follow the Sema domains and are composed of one or more domains. They have, for example, in these C-terminal amino acid sequences transmembrane domains (TM), immunoglobulin-like domains (Ig) (constant part of the immunoglobulin), cytoplasmic sequences (CP), processing signals (P) (for example having the consensus sequence (RXR) where R is the amino acid arginine and X is any amino acid) and/or hydrophilic C termini (HPC). The semaphorins disclosed to date can be divided on the basis of the differences in the domain structure in the C terminus into 5 different subgroups (I to V):
ISecreted, without other domains (for exampleORF-A49)IIIgSecreted (without transmembrane domain) for exampleAHV-Sema)IIIIg, TM, CPMembrane-anchored with cytoplasmic sequence(for example CD100)IVIg, (P), HPCSecreted with hydrophilic C terminus (for exampleH-Sema III, M-SemaD, collapsin-1)VIg, TM, CPMembrane-anchored with C-terminal 7thrombospondin motif (for example M-SemaF and G)
A receptor or extracellular ligand for semaphorins has not been described to date. Intracellular, heterotrimeric GTP-binding protein complexes have been described in connection with semaphorin-mediated effects. One component of these protein complexes which has been identified in chickens is called CRMP (collapsin response mediator protein) and is presumed to be a component of the semaphorin-induced intracellular signal cascade (Goshima et al. (1995) Nature 376: 509-514). CRMP62, for example, has homology with unc-33, a nematode protein which is essential for directed growth of axons. A human protein with 98% amino acid identity with CRMP62 is likewise known (Hamajima et al. (1996) Gene 180: 157-163). Several CRMP-related genes have likewise been described in rats (Wang et al. (1996) Neurosci. 16: 6197-6207).
The secreted or transmembrane semaphorins convey repulsive signals for growing nerve buds. They play a part in the development of the central nervous system (CNS) and are expressed in particular in muscle and nerve tissues (Kolodkin et al. (1993); Luo et al. (1993) Cell 75:217-227).
Pronounced expression of M-SemaG has been observed not only in the CNS but also in cells of the lymphatic and hematopoietic systems, in contrast to the closely related M-SemaF {Furuyima et al. (1996) J. Biol. Chem. 271: 33376-33381}.
Recently, two other human semaphorins have been identified, H-Sema IV and H-Sema V, specifically in a region on chromosome 3p21.3, whose deletion is associated with various types of bronchial carcinomas. H-Sema IV {Roche et al. (1996), Xiang et al. (1996), Sekido et al. (1996)} is about 50% identical at the amino acid level with M-SemaE, whereas H-Sema V {Sekido et al. (1996)} is the direct homolog of M-SemaA (86% amino acid identity). Since these genes (H-Sema IV and V) were found during DNA sequencing projects on the deleted 3p21.3 loci, the complex intron-exon structure of these two genes is known. Both genes are expressed in various neuronal and non-neuronal tissues.
Likewise only recently, the cellular surface molecule CD100 (human), expressed and induced on activated T cells, has been identified as a semaphorin (likewise listed in Table 1). It assists interaction with B cells via the CD40 receptor and the corresponding ligand CD40L. CD100 is a membrane-anchored glycoprotein dimer of 150 kd (kilodaltons). An association of the intracytoplasmic C-terminus of CD100 with an as yet unknown kinase has been described {Hall et al. (1996)}. This means that CD100 is the first and to date only semaphorin whose expression in cells of the immune system has been demonstrated.
In the “transforming genes of rhadinoviruses” project, the complete genome of alcelaphine herpesvirus Type 1 (AHV-1) has been cloned and sequenced {Ensser et al. (1995)}. AHV-1 is the causative agent of malignant catarrhal fever, a disease of various ruminants which is associated with a lymphoproliferative syndrome and is usually fatal. On analysis, an open reading frame was found, at one end of the viral genome, having remote but significant homology with a gene of vaccinia-virus (ORF-A39 corresponds to VAC-A39 in Ensser et al. (1995) J. Gen. Virol. 76:1063-1067) which has been assigned to the semaphorin gene family. Whereas the AHV-1 semaphorin (AHV-Sema) has a well-conserved semaphorin structure, the poxvirus genes (ORF-A39 and ORF-A39-homologous, see Table 1) have C-terminal truncations, i.e. the conserved Sema domain is present in them only incompletely.
Databank comparison of the found AHV-Sema with dbEST (EST (expressed sequence tags) databank (db)) provided in each case 2 EST sequences from 2 independent cDNA clones from human placenta (accession numbers H02902, H03806 (clone 151129), accession numbers R33439 and R33537 (clone 135941)). These display distinctly greater homology with AHV-1 semaphorin than with the neuronal semaphorins hitherto described.